Polycystin2 function in Kidney Development

多囊蛋白2在肾脏发育中的功能

• 批准号: 7111712
• 负责人: IAIN A. DRUMMOND
• 金额: $ 25.22万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7111712
• 关键词: alternatives to animals in research; biological signal transduction; calcium channel; developmental genetics; disease /disorder model; epithelium; gene expression; genetic regulation; intermolecular interaction; kidney; molecular pathology; nephrogenesis; polycystic kidney; protein localization; protein structure function; zebrafish

Autosomal dominant polycystic kidney disease is caused primarily by mutations in two genes, Pkd1 and Pkd2, which function together as a calcium channel complex. Subcellular localization studies suggest that the polycystin2 protein may act in the apical cell membrane associated with cilia or in internal cell membranes as a calcium release channel. However, it is currently not known where polycystin2 channel activity acts in epithelia or which cellular signaling systems may impinge on polycystin2 function. Disruption of zebrafish polycystin2 gene expression using antisense morpholino oligos results in rapid kidney cyst development, randomized organ laterality, hydrocephalus, and body axis curvature. These defects are rescued by co-injection of the human pkd2 mRNA. In Aim 1. we propose using the zebrafish as an in vivo system for structure function analysis of polycystin2. We hypothesize that specific amino acid motifs in polycystin2 target it to its cellular site of action. By disrupting these either in the endogenous zebrafish polycystin2 or in rescuing human polycystin2 mRNAs we will test whether subcellular localization affects the cellular function of polycystin2. In Aim 2. we hypothesize that polycystin 2, acting as a calcium channel, mediates the effects of physical forces on the epithelium. We will measure calcium responses in isolated kidney tubules that lack polycystin2 function or that express altered pkd2 alleles. Finally we propose that mutations in polycytin2 interacting proteins or downstream mediators may effect signaling or function of polycystin2. In Aim 3. we outline a plan to identify cellular components that interact with polycystin2 by screening for dominant suppressors of the zebrafish polycystin2 phenotype. This proposal exploits unique advantages of the zebrafish as a model organism to more fully explore the function of polycystin2 in vivo and further our understanding of the cellular mechanisms of autosomal dominant polycystic kidney disease.

常染色体显性多囊性肾脏疾病主要是由两个基因PKD1和PKD2中的突变引起的，它们可以作为钙通道复合物一起起作用。亚细胞定位研究表明，PolycyStin2蛋白可以在与纤毛或内部细胞膜相关的根尖细胞膜中作用，作为钙释放通道。但是，目前尚不清楚polycyStin2通道活性在上皮中起作用或哪种细胞信号系统可能影响polycyStin2功能。 使用反义莫诺利诺寡糖杀伤斑马鱼polycystin2基因表达会导致快速肾脏囊肿的发育，随机器官横向性，脑积水和身体轴曲率。这些缺陷是通过共同注射人类PKD2 mRNA挽救的。在AIM 1中。我们建议将斑马鱼用作PolycyStin2结构函数分析的体内系统。我们假设PolycyStin2中的特定氨基酸基序将其靶向其细胞作用部位。通过在内源性斑马鱼polycystin2中破坏它们，或者在营救人Polycystin2 mRNA中，我们将测试亚细胞定位是否影响PolycyStin2的细胞功能。在AIM 2中。我们假设Polycystin 2充当钙通道， 介导物质对上皮的影响。我们将测量缺乏多囊菌功能或明显改变PKD2等位基因的孤立肾小管中的钙反应。最后，我们提出，聚囊蛋白相互作用蛋白或下游介质中的突变可能影响polycystin2的信号传导或功能。在AIM 3中。我们概述了一个计划，通过筛选斑马鱼PolycyStin2表型的显性抑制剂来鉴定与PolycyStin2相互作用的细胞成分。 该提案利用斑马鱼作为模型生物的独特优势，以更充分地探索Polycystin2在体内的功能，并进一步了解我们对常染色体显性多囊肾脏疾病的细胞机制的理解。